Sunday, 16 August 2009

During the first two terms of my final year at university I took on a dry project based on bacterial genetics. In retrospect this was quite a cheap move since it avoids so many of the problems involved in normal lab work, but in my defence it was the project that interested me most and I really wanted some computing experience before making PhD applications.My work was based on bacterial endosymbionts of insects, which have been of recent interest due to their extremely small genomes. Bacteria in these symbiotic relationships are given a protected, nutrient-rich environment and in return they allow insects to survive on unbalanced diets by synthesising scarce biomolecules.

Primary endosymbiotic bacteria live their entire lives inside insects and are vertically transmitted from generation to generation, a process that leads to coevolution between the bacteria and the insect. One of the results of this coevolution was major changes to the original bacterial genome, which contained many genes that are essential for free-living bacteria but are unnecessary for life within an insect. Consequently, common features of endosymbiotic genomes compared to those of free-living bacteria are severe gene loss, genome compaction and skewing of GC content.

Electron micrograph showing bacteriocytes taken from P. venusta

1 – Bacteriocyte; 2 – C. ruddii; 3 – Unidentified electron-dense mass

My project focused on Carsonella ruddii, the only bacterial endosymbiont of the psyllid, Pachypsylla venusta. It was hailed as the smallest bacterial genome chracterised when it was sequenced in 2006 and still holds that record. Its genome contains only 182 ORFs, less than 3% intergenic DNA and has a GC content of 16.5%. The bacteria appears to be provided with many nutrients by its host and its metabolism has been reduced to a few pathways: ATP synthesis, a section of the pentose phosphate pathway and biosynthesis of certain amino acids.

The early stages of my project involved a reannotation of the C. ruddii genome followed by a sequence-based functional analysis of its metabolic enzymes. Using the enzymes deemed functional in this analysis I built an updated model of the C. ruddii metabolism which could be divided into six pathways involved in amino acid biosynthesis, five of which were incomplete. The only fully intact pathway led to the production of isoleucine and valine. These are both essential amino acids for insects and are severely under-represented in the adult psyllid diet.

Four of the incomplete amino acid pathways were missing only one reaction and the conservation of the rest of each of the pathways suggested that they might still be functional in C. ruddii. The ‘missing’ reactions might occur spontaneously under some conditions or could be catalysed by unidentified enzymes. For three of these four missing reactions I found an example in the literature of a different bacterial endosymbiont which had lost that reaction but had retained the rest of the pathway. This seemed to suggest that the enzymes catalysing these reactions might be expendable and subject to loss during genome reduction in endosymbionts. Based on this and some other evidence from similar situations in endosymbionts I predicted that these pathways are probably functional in C. ruddii and that its main role symbiotic role is to provide the psyllid with essential amino acids.

The fourth of these incomplete pathways was the most interesting because I was unable to locate another endosymbiont which was missing the same reaction. The reaction was catalysed by the product of a gene, AS, which was present on the C. ruddii genome but which I had labelled as a pseudogene during functional analysis. Although it’s difficult to conclusively say that an enzyme is inactive solely by sequence analysis, multiple alignments showed that this copy of AS was extensively degraded and was missing both of its key catalytic residues as well as its substrate binding residues. However, later in the project when I was scanning an EST set taken from the insect host of C. ruddii I located another copy of AS which also had bacterial origin but which was not present on the C. ruddii genome. Sequence analysis showed that this version of AS seemed to be active and could potentially fill the gap in the pathway.

Where did this copy of AS originate from? It aligned well with the version of AS from P.aeruginosa and appeared to have a bacterial origin but was not found on the C. ruddii genome or the psyllid mitochondrial genome, both of which have been sequenced. Several lines of evidence ruled out the presence of a second bacterial endosymbiont in this symbiosis and since no plasmids had been reported during DNA sequencing of C. ruddii the source of this sequence appeared to be the nuclear genome of P. venusta itself.The presence of this bacterial sequence in the eukaryotic genome suggests that LGT may have taken place between a bacterial genome and the insect nuclear genome. This would be one explanation for the fact that C. ruddii has only 182 ORFs, which is significantly lower than the predicted minimal bacterial genome. However, it is also possible that C. ruddii uses mitochondrial proteins to survive and so LGT is not the only explanation for the low ORF count.

This was my favourite line of investigation during my project but the symbiosis between C. ruddii and P. venusta had many more interesting features that I read about over the year. One of the questions I got in my viva was whether C. ruddii should be labelled as a bacterium or an organelle. I think this question is only really important when considering a minimal bacterial genome and if C. ruddii does turn out to be importing essential proteins from elsewhere then I think that the label organelle is definitely more appropriate. However, the definition of an organelle doesn't seem to be well-established and so whether or not C. ruddii really does have the smallest bacterial genome is a matter of opinion.

Contributors

We are geneticists and biochemists, alumni of the University of York (2009), now doing PhDs at the Universities of Cambridge, Leeds, Oxford and Vermont. We aim to bring to your attention interesting science, whether it is making headlines or not, referencing the original peer-reviewed research as often as possible.